This invention relates to oral compositions and immunogenic compositions for use in the suppression of the pathogenic effects of the bacterium Porphyromonas gingivalis associated with periodontal disease and cardiovascular disease. It also relates to diagnostic tests for the presence of Porphyromonas gingivalis in subgingival plaque samples and specific antibodies against P. gingivalis antigens. The compositions comprise proteins, peptides or oligopeptides or peptide chimeras of specific antigens of Porphyromonas gingivalis. Also disclosed are methods for preparing the antigens, peptide components and peptide chimeras using recombinant DNA and/or biochemical techniques. Related thereto, disclosed are the DNA sequences encoding the specific antigens, and recombinant vectors useful in directing the expression of antigen constructs containing major epitopes. Also disclosed are host cells transformed with such recombinant vectors. The proteins, peptides, oligopeptides and peptide chimeras are useful as immunogens in formulations for use in raising an immune response and can be used to generate protein-specific and peptide-specific antisera useful for passive immunization and as reagents for diagnostic assays. The nucleotide sequences disclosed provide for the synthesis of corresponding oligonucleotides which can be used as reagents in diagnostic assays directed to the detection of P. gingivalis genetic material and incorporated into expression vectors for use as genetic vaccine formulations.
Periodontal diseases are bacterial-associated inflammatory diseases of the supporting tissues of the teeth and range from the relatively mild form of gingivitis, the non-specific, reversible inflammation of gingival tissue to the more aggressive forms of periodontitis which are characterised by the destruction of the tooth""s supporting structures. Periodontitis is associated with a subgingival infection of a consortium of specific Gram-negative bacteria that leads to the destruction of the periodontium and is a major public health problem. One bacterium that has attracted considerable interest is Porphyromonas gingivalis as the recovery of this microorganism from adult periodontitis lesions can be up to 50% of the subgingival anaerobically cultivable flora, whereas P. gingivalis is rarely recovered, and then in low numbers, from healthy sites. A proportional increase in the level of P. gingivalis in subgingival plaque has been associated with an increased severity of periodontitis and eradication of the microorganism from the cultivable subgingival microbial population is accompanied by resolution of the disease. The progression of periodontitis lesions in non-human primates has been demonstrated with the subgingival implantation of P. gingivalis. These findings in both animals and humans suggest a major role for P. gingivalis in the development of adult periodontitis. The presence of P. gingivalis in atheromatous plaques has also been associated with the development of cardiovascular disease.
P. gingivalis is a black-pigmented, anaerobic, proteolytic Gram-negative rod that obtains energy from the metabolism of specific amino acids. The microorganisms has an absolute growth requirement for iron, preferentially in the form of heme or its Fe(III) oxidation product hemin and when grown under conditions of excess hemin is highly virulent in experimental animals. A number of virulence factors have been implicated in the pathogenicity of P. gingivalis including the capsule, adhesins, cytotoxins and extracellular hydrolytic enzymes. In order to develop an efficacious and safe vaccine to prevent P. gingivalis colonisation it is necessary to identify protein antigens that are involved in virulence that have utility as immunogens to generate neutralising antibodies.
The present inventors purified and characterised four major P. gingivalis antigens using serum from a healthy subject that harboured P. gingivalis in subgingival plaque as shown by DNA probe analysis. The antigens (Ag1, Ag2, Ag3 and Ag4) are listed below.
Accordingly in a first aspect the present invention consists in a composition for use in raising an immune response directed against Porphyromonas gingivalis, the composition including a suitable adjuvant and/or acceptable carrier and one substantially purified P. gingivalis immunogen, the immunogen being selected from the group consisting of Antigen 1, Antigen 2, Antigen 3, Antigen 4 and epitope containing fragments thereof. Optionally, the composition may further include at least one additional purified P. gingivalis immunogen, the immunogen being selected from the group consisting of Antigen 1, Antigen 2, Antigen 3, Antigen 4 and epitope containing fragments thereof.
In a second aspect, the present invention consists in a substantially purified P. gingivalis antigen or epitope containing fragment thereof, wherein antigen has an internal amino acid sequence:
DLENKGEATLLVTFGSSYKAPRETYAKIEKTFAAAYPDQR (SEQ ID NO:1). It is preferred that the antigen has an amino acid sequence as shown in FIG. 1 (SEQ ID NOS:5 and 6).
In a third aspect, the present invention consists in a substantially purified P. gingivalis antigen or epitope containing fragment thereof, wherein antigen has an internal amino acid sequence:
DNPDENPLEGDITQTHTEKYVLAED (SEQ ID NO:2).
In a fourth aspect, the present invention consists in a substantially purified P. gingivalis antigen or epitope containing fragment thereof, wherein antigen has an internal amino acid sequence:
DVLLLDVTPLSLGIETMGGVMTYLIDANTTIPKLK (SEQ ID NO:3). It is preferred that the antigen includes an amino acid sequence encoded by the open reading frame of the clone deposited with AGAL under accession No. NM 97/04974 which hybridises with degenerate probes corresponding to the amino acid sequence DVLLLDVTPLSLGIETMGGVMTYLIDANTTIPKLK (SEQ ID NO:3).
In a fourth aspect, the present invention consists in a substantially purified P. gingivalis antigen antigen or epitope containing fragment thereof, wherein antigen has an internal amino acid sequence: VYNASISAVGNTSAIDPVVQIIHHN (SEQ ID NO:4).
In other aspects, the present invention consists in nucleotide sequences encoding Ag1, Ag2, Ag3 and Ag4 and probes which hybridise to these sequences.
The nucleotide sequence encoding Ag1 and deduced amino acid sequence of the haeme receptor protein Ag1 is shown in FIG. 1. The disclosure of the nucleotide sequence includes within its scope degeneracy equivalents and subsequences coding for amino acid sequences corresponding to antigenic determinants of P. gingivalis W50.
A clone containing nucleotide sequence from Ag3, DnaK clone #6, has been deposited under the terms of the Budapest Treaty with Australian Government Analytical Laboratories, 1 Suakin Street, Pymble, NSW Australia on Mar. 25, 1997 and has been accorded accession No. NM 97/04974. Accordingly further nucleotide sequence for this antigen can be obtained by accessing this deposit. Access to this deposit is available under the terms and conditions of the Budapest Treaty. Where applicable access to this deposit is to be limited to independent experts (EPC Rule 28(4). AU Reg. 3.25(3)).
In another aspect the present invention consists in antibodies raised against the antigens of the present invention.
Antibodies against the antigens can be used in oral compositions such as toothpaste and mouthwash to neutralise the antigens and thus prevent disease. Antigen-specific antibodies can also be used for the early detection of P. gingivalis in subgingival plaque samples by a diagnostic assay. A vaccine based on these antigens and suitable adjuvant delivered by nasal spray, orally or by injection to produce a specific immune response against these antigens thereby reducing colonisation and virulence of P. gingivalis and thereby preventing or reducing disease. The antigen proteins and antigen peptides (herein termed xe2x80x9cpeptidesxe2x80x9d) and antigen oligopeptides (herein termed xe2x80x9coligopeptidesxe2x80x9d) and antigen chimeric peptides containing epitopes of one antigen fused with the epitopes of another (herein termed xe2x80x9cchimeric peptidesxe2x80x9d) thereof, of the present invention may be used as immunogens in prophylactic and/or therapeutic vaccine formulations; or as an antigen in diagnostic immunoassays directed to detection of P. gingivalis infection by measuring an increase in serum titer of P. gingivalisxe2x80x94specific antibody. Also antigen protein, peptides, oligopeptides and chimeric peptides of the present invention may be used to generate antigen-specific antibody which may be useful for passive immunotherapy and as reagents for diagnostic assays directed to detecting the presence of P. gingivalis in clinical specimens such as subgingival plaque samples. Peptides, oligopeptides or chimeric peptides can be obtained by chemical synthesis, purification from P. gingivalis cultures, or produced from recombinant vector expression systems using the nucleic acid sequences disclosed herein.
Accordingly, in other aspects the invention provides oral compositions including toothpastes and mouthwashes which include antibodies raised against any one or a combination of antigens Ag1, Ag2, Ag3 and Ag4.
In another aspect the invention provides a method of early detection of P. gingivalis comprising a diagnostic assay involving the use of antibodies raised against any one or a combination of antigens Ag1, Ag2, Ag3 and Ag4.
In another aspect, the invention provides a method for the detection of P. gingivalis infection comprising the measure of an increase in serum titer to any one of the P. gingivalis antigens as herein described.
Other aspects of the present invention are directed to the construction of novel DNA sequences involving antigen constructs and vectors including plasmid DNA, and viral DNA such as human viruses, animal viruses, insect viruses, or bacteriophages which can be used to direct the expression of antigen protein, peptides, oligopeptides or chimeric peptides in appropriate host cells from which the expressed protein or peptides may be purified.
Another aspect of the present invention provides methods for molecular cloning of the genes encoding the antigens Ag1, Ag2, Ag3 and Ag4, and gene fragments encoding antigen peptides or oligopeptides or chimeric peptides.
The nucleic acid sequences of the present invention can be used in molecular diagnostic assays for P. gingivalis genetic material through nucleic acid hybridization, and including the synthesis of antigen sequence-specific oligonucleotides for use as primers and/or probes in amplifying, and detecting amplified, nucleic acids. Additionally, antigen protein, peptides, oligopeptides, chimeric peptides and antigenic constructs containing epitopes can be used as immunogens in prophylactic and/or therapeutic vaccine formulations against pathogenic strains of P. gingivalis, whether the immunogen is chemically synthesized, purified from P. gingivalis, or purified from a recombinant expression vector system. Alternatively, the genes encoding the antigens, or one or more gene fragments encoding peptides or oligopeptides or chimeric peptides, may be incorporated into a bacterial or viral vaccine comprising recombinant bacteria or virus which is engineered to produce one or more immunogenic epitopes of each antigen by itself, or in combination with immunogenic epitopes of other antigens or from other pathogenic microorganisms. In addition, the genes encoding the antigens or one or more gene fragments encoding peptides or oligopeptides or chimeric peptides, operatively linked to one or more regulatory elements, can be introduced directly into humans to express protein, peptide, oligopeptides or chimeric peptides relating to the antigens to elicit a protective immune response. A vaccine can also be based upon a recombinant component of a mutated antigen incorporated into an appropriate vector and expressed in a suitable transformed host (e.g. E. coli, Bacillus subtilis, Saccharomyces cerevisiae, COS cells, CHO cells and HeLa cells) containing the vector. The vaccine can be based on an intra-oral recombinant bacterial vaccine, where the recombinant bacterium expressing antigen is a commensal inhabitant of the oral cavity. Unlike whole P. gingivalis cells or other previously prepared antigens, the four antigens described herein are safe and effective antigens for the preparation of a vaccine for the prevention of P. gingivalis-associated periodontal disease.
In yet another aspect the present consists in a composition for use in raising an immune response directed against Porphyromonas gingivalis, the composition including a suitable adjuvant and/or acceptable carrier and a DNA molecule including a sequence encoding one P. gingivalis immunogen, the immunogen being selected from the group consisting of Antigen 1, Antigen 2, Antigen 3, Antigen 4 and epitope containing fragments thereof. P. gingivalis has an absolute growth requirement for Fe which it prefers in the form of haeme. As such, Ag1 is of particular interest as neutralisation of this haeme receptor by specific antibodies would prevent haeme uptake and therefore growth and virulence. P. gingivalis grown haeme-limited is less virulent in animal models.
Fimbriae are thin, filamentous structures that either completely cover the cell or are polar. There are at least two fimbrial types recognized: those involved in the transfer of genetic material by the formation of conjugation bridges, and those involved in adherence to soft and hard tissues. The fimbriae involved in conjugation are referred to as sex pili. These pili have specific receptors for attachment to a genetically compatible recipient bacterium. The second fimbrial type, the type specific or common pili are involved in eubacterial coaggregation and adherence to eukaryotic cells and often play an important role in prevention of phagocytosis and the invasion of host tissue. In the Enterobacteriaceae, the fimbriae consist of repeating subunit proteins of approximately 17 to 21 kDa. Minor proteins are also part of the fimbriae structure. The specific fimbrial binding proteins (adhesins) are often 28 to 31 kDa and located at the tip or periodically along the length of the fimbriae.
Yoshimura et al. were amongst the first to demonstrate the presence of fimbriae on P. gingivalis and they purified a 43 kDa fimbrilin subunit which has no amino acid sequence homology with fimbrilins from other Gram-negative bacteria. Genco and coworkers have shown that the 43 kDa fimbrial protein and synthetic peptides corresponding to the C-terminal end of the fimbrilin reduced adherence of P. gingivalis 381 to saliva-coated hydroxyapatite. In a subsequent study they showed that a recombinant fimbrilin binds specifically to statherin and proline-rich proteins of saliva. Immunisation of rats with the 43 kDa protein protected against periodontal tissue destruction induced by infection with P. gingivalis 381. Further, an isogenic mutant of P. gingivalis 381 with the fimA gene, that encodes the 43 kDa fimbrilin, insertionally inactivated was significantly less able to produce periodontal tissue destruction in the rat model when compared with the wild-type strain. In this study it should be noted however, that the fimA mutant still did produce greater periodontal tissue destruction than occurred in the sham infected animals. The fimA mutant was unimpaired in its ability to agglutinate red blood cells, coaggregate with other oral bacteria although binding to saliva-coated hydroxyapatite was reduced. In an independent study Hamada et al. also produced a fimA mutant of P. gingivalis ATCC 33277 by homologous recombination of an insertionally inactivated gene and noted that although the mutant failed to express long (0.5 to 1.0 xcexcm) fimbriae, thin, short fimbrial structures could still be observed by electron microscopy suggesting the presence of a second fimbrial type. The identification of the 43 kDa fimbrilin and the virulence and immunisation studies related to the 43 kDa protein have been conducted with the P. gingivalis strains 381 and ATCC 33277. These strains are classified as non-invasive and are considered to be less virulent than invasive strains based on the infective process in animal models. Non-invasive strains produce a localised abscess at the challenged site, whereas invasive strains at the same inoculum spread to distant sites and produce multiple abscesses. Further, Sundqvist et al. showed that the non-invasive strains (381 and ATCC 33277) were phagocytosed and killed by polymorphonuclear leukocytes to a high extent whereas the invasive strains W50 and W83 were poorly phagocytosed and killed. It is interesting to note that Naito et al. suggest that there is a difference in the fimbriae of invasive and non-invasive P. gingivalis strains. These workers found that the fimbriae of non-invasive strains bound to collagen-coated hydroxyapatite (HA) in high numbers whereas the fimbriae prepared from invasive strains bound to collagen-coated HA weakly. The P. gingivalis invasive strains W50, W83 and AJW5 are highly virulent in animal models but do no express the 43 kDa fimbrilin as shown by immunocytochemistry and Western blot analysis. However, on fine negative staining, W50 and W83 are fimbriated although less densely than other strains. It appears that W50 and W83 possess inactive fimA genes accounting for the lack of the 43 kDa fimbrilin however these strains are still virulent and invasive despite lacking the 43 kDa fimbrilin.
Ag2 is the second fimbrial type or a major adhesin of P. gingivalis. As part of a study to purify and characterise cell surface protein antigens of P. gingivalis W50 we purified a 30 kDa fragment of the 46 kDa fimbrial protein (Ag2) that was seroreactive with serum from a healthy subject that harboured P. gingivalis subgingivally as shown by DNA probe analysis. The internal amino acid sequence of the 30 kDa fragment showed considerable homology (48% identity) to a fimbrial protein of Dichelobacter (formerly Bacteroides) nodosus.
P. gingivalis 30 kDa fragment DNPDENPLEGDITQTHTEKYVLAED (SEQ ID NO:2) . . . D. nodosus fimbrial protein KGPDANPASGVVGNKDTGKYVLAEI . . . (SEQ ID NO:7)
The D. nodosus fimbrial protein is classified as a type-IV or mePhe pilin which is a common fimbrial type of a group of Gram-negative bacteria including Bacteroides spp., Neisseria gonorrhoeae, Neisseria meningitidis, Acinetobacter calcoaceticus, Eikenella corrodens, Moraxella bovis, Moraxella nonliquefaciens and several species of Pseudomonas including P. aeruginosa (Elleman, 1988). The P. gingivalis 30 kDa fragment exhibits the highest homology with the conserved amino acyl residues of the central domain of the D. nodosus A-set or Class I fimbriae (including the serotypes A, B, C, E, F, and G). A characteristic of the type-TV fimbriae is that they adhere to eukaryotic cells and agglutinate red blood cells. It is interesting to note that the conserved hexapeptide motif-KYVLAE- (SEQ ID NO:8) which is also present in the P. gingivalis 46 kDa fimbrial protein has been localised to, or near to, the receptor binding site of gonococcal pili since antisera to these residues prevents bacterial attachment to eukaryotic cells even by heterologous pili and precipitates larger peptide fragments which bind to eukaryotic cells. It has been suggested that the conserved residues of the central domain when juxtaposed form a cleft which specifically interacts with the carbohydrate moieties of surface glycoproteins of eukaryotic cells.
D. nodosus is the aetiological agent of the contagious disease of sheep, interdigital dermatitis or footrot, and the type-IV fimbriae are the major serological and immunoprotective virulence factors. Footrot vaccines have evolved from simple bacterins to highly specific recombinant DNA fimbrial vaccines. The initial whole cell vaccines were unsuccessful due to the short duration of immunity and incorporation of limited serotypes. A number of antigens were examined and the major protective immunogen was the type-IV fimbrial subunit protein. Monovalent vaccines based on recombinant fimbriae are omnipotent inducing long lasting immunity. The homology between the P. gingivalis 46 kDa fimbrial protein and the D. nodosus immunoprotective fimbriae indicate that the P. gingivalis protein (Ag2) would have application in diagnostic and immunoprophylactic products for P. gingivalis-related periodontitis.
The heat shock or stress response of cells is a homeostatic mechanism that enables cells to survive environmental stresses such as temperature elevation that can result in denaturation of cellular proteins. The DnaK family of proteins bind to denatured and incorrectly folded proteins and facilitate refolding to the original conformation and function. The DnaK or Heat Shock Protein (HSP) 70 is a highly conserved molecular chaperonin common to bacterial and eukaryotic cells comprising 1-5% of the constitutive cellular protein, with 15% of DnaK in E. coli being associated with vesicles. During stress DnaK can be overexpressed to constitute up to 30% of total cellular protein making this protein an ideal candidate for a sensitive immunodiagnostic test. Specific diagnostic tests for leprosy and tuberculosis have been developed based on the respective DnaK protein homologues. All species homologues of DnaK are highly conserved in the N-terminal half of the protein with the species-specific regions of the molecule in the C-terminal half. The P. gingivalis DnaK homolgue (Ag3) therefore would have application in diagnostic and immunoprophylactic products for P. gingivalis-related periodontitis.
The four antigens identified (Ag1, Ag2, Ag3 and Ag4) are of particular interest for diagnostics and neutralisation by passive immunity through oral compositions containing neutralising antibodies and by vaccine development. In particular for the development of an intra-oral recombinant bacterial vaccine, where the recombinant bacterium expressing the antigens is a genetically engineered commensal inhabitant of the oral cavity. The superiority of these four antigens to prior disclosed P. gingivalis antigens, is that these are major virulence-associated factors and contain conserved epitopes on invasive strains making them ideal for the development of diagnostic and immunoprophylactic products.
The present invention will now be described in detail with reference to the following particularly preferred embodiments which are not limiting to the invention but representative of the methods of performing certain aspects of the invention.
The four antigens Ag1, Ag2, Ag3 and Ag4 can be purified from P. gingivalis cells by chloroform extraction followed by anion exchange, gel filtration and reversed-phase chromatography. The purified antigens are then used to generate polyclonal or monoclonal antibodies using standard techniques. The animals used for antibody generation can be mice, rabbits, goats, chickens, sheep, horses, cows etc. When a high antibody titre against the antigens is detected by immunoassay the animals are bled or eggs or milk are collected and the serum prepared and/or antibody purified using standard techniques or monoclonal antibodies produced by fusing spleen cells with myeloma cells using standard techniques. The antibody (immunoglobulin fraction) may be separated from the culture or ascites fluid, serum, milk or egg by salting out, gel filtration, ion exchange and/or affinity chromatography, and the like, with salting out being preferred. In the salting out method the antiserum or the milk is saturated with ammonium sulphate to produce a precipitate, followed by dialyzing the precipitate against physiological saline to obtain the purified immunoglobulin fraction with the specific antibody. The preferred antibody is obtained from the equine antiserum and the bovine antiserum and milk. In this invention the antibody contained in the antiserum and milk obtained by immunising the animal with the antigens is blended into the oral composition. In this case the antiserum and milk as well as the antibody separated and purified from the antiserum and milk may be used. Each of these materials may be used alone or in combination of two or more. Antibodies can be used in oral compositions such as toothpaste and mouthwash to neutralise P. gingivalis and thus prevent disease. The antibodies can also be used for the early detection of P. gingivalis in subgingival plaque samples by a chairside Enzyme Linked Immunosorbent Assay (ELISA).
For oral compositions it is preferred that the amount of the above antibodies administered is 0.0001-50 g/kg/day and that the content of the above antibodies is 0.0002-10% by weight preferably 0.002-5% by weight of the composition. The oral composition of this invention which contains the above-mentioned serum or milk antibody may be prepared and used in various forms applicable to the mouth such as dentifrice including toothpastes, toothpowders and liquid dentifrices, mouthwashes, troches, chewing gums, dental pastes, gingival massage creams, gargle tablets, dairy products and other foodstuffs. The oral composition according to this invention may further include additional well known ingredients depending on the type and form of a particular oral composition.
In certain highly preferred forms of the invention the oral composition may be substantially liquid in character, such as a mouthwash or rinse. In such a preparation the vehicle is typically a water-alcohol mixture desirably including a humectant as described below. Generally, the weight ratio of water to alcohol is in the range of from about 1:1 to about 20:1. The total amount of water-alcohol mixture in this type of preparation is typically in the range of from about 70 to about 99.9% by weight of the preparation. The alcohol is typically ethanol or isopropanol. Ethanol is preferred. The pH of such liquid and other preparations of the invention is generally in the range of from about 4.5 to about 9 and typically from about 5.5 to 8. The pH is preferably in the range of from about 6 to about 8.0, preferably 7.4. The pH can be controlled with acid (e.g. citric acid or benzoic acid) or base (e.g. sodium hydroxide) or buffered (as with sodium citrate, benzoate, carbonate, or bicarbonate, disodium hydrogen phosphate, sodium dihydrogen phosphate, etc).
Other desirable forms of this invention, the oral composition may be substantially solid or pasty in character, such as toothpowder, a dental tablet or a dentifrice, that is a toothpaste (dental cream) or gel dentifrice. The vehicle of such solid or pasty oral preparations generally contains dentally acceptable polishing material. Examples of polishing materials are water-insoluble sodium metaphosphate, potassium metaphosphate, tricalcium phosphate, dihydrated calcium phosphate, anhydrous dicalcium phosphate, calcium pyrophosphate, magnesium orthophosphate, trimagnesium phosphate, calcium carbonate, hydrated alumina, calcined alumina, aluminum silicate, zirconium silicate, silica, bentonite, and mixtures thereof. Other suitable polishing material include the particulate thermosetting resins such as melamine-, phenolic, and urea-formaldehydes, and cross-linked polyepoxides and polyesters. Preferred polishing materials include crystalline silica having particle sized of up to about 5 microns, a mean particle size of up to about 1.1 microns, and a surface area of up to about 50.000 cm2/gm., silica gel or colloidal silica, and complex amorphous alkali metal aluminosilicate.
When visually clear gels are employed, a polishing agent of colloidal silica, such as those sold under the trademark SYLOID as Syloid 72 and Syloid 74 or under the trademark SANTOCEL as Santocel 100, alkali metal alumino-silicate complexes are particularly useful since they have refractive indices close to the refractive indices of gelling agent-liquid (including water and/or humectant) systems commonly used in dentifrices.
Many of the so-called xe2x80x9cwater insolublexe2x80x9d polishing materials are anionic in character and also include small amounts of soluble material. Thus, insoluble sodium metaphosphate may be formed in any suitable manner as illustrated by Thorpe""s Dictionary of Applied Chemistry, Volume 9, 4th Edition, pp. 510-511. The forms of insoluble sodium metaphosphate known as Madrell""s salt and Kurrol""s salt are further examples of suitable materials. These metaphosphate salts exhibit only a minute solubility in water, and therefore are commonly referred to as insoluble metaphosphates (IMP).
There is present therein a minor amount of soluble phosphate material as impurities, usually a few percent such as up to 4% by weight. The amount of soluble phosphate material, which is believed to include a soluble sodium trimetaphosphate in the case of insoluble metaphosphate, may be reduced or eliminated by washing with water if desired. The insoluble alkali metal metaphosphate is typically employed in powder form of a particle size such that no more than 1% of the material is larger than 37 microns.
The polishing material is generally present in the solid or pasty compositions in weight concentrations of about 10% to about 99%. Preferably, it is present in amounts from about 10% to about 75% in toothpaste, and from about 70% to about 99% in toothpowder. In toothpastes, when the polishing material is silicious in nature, it is generally present in amount of about 10-30% by weight. Other polishing materials are typically present in amount of about 30-75% by weight.
In a toothpaste, the liquid vehicle may comprise water and humectant typically in an amount ranging from about 10% to about 80% by weight of the preparation. Glycerine, propylene glycol, sorbitol and polypropylene glycol exemplify suitable humectants/carriers. Also advantageous are liquid mixtures of water, glycerine and sorbitol. In clear gels where the refractive index is an important consideration, about 2.5-30% w/w of water, 0 to about 70% w/w of glycerine and about 20-80% w/w of sorbitol are preferably employed.
Toothpaste, creams and gels typically contain a natural or synthetic thickener or gelling agent in proportions of about 0.1 to about 10, preferably about 0.5 to about 5% w/w. A suitable thickener is synthetic hectorite, a synthetic colloidal magnesium alkali metal silicate complex clay available for example as Laponite (e.g. CP, SP 2002, D) marketed by Laporte Industries Limited. Laponite D is, approximately by weight 58.00% SiO2, 25.40% MgO, 3.05% Na2O, 0.98% Li2O, and some water and trace metals. Its true specific gravity is 2.53 and it has an apparent bulk density of 1.0 g/ml at 8% moisture.
Other suitable thickeners include Irish moss, iota carrageenan, gum tragacanth, starch, polyvinylpyrrolidone, hydroxyethylpropylcellulose, hydroxybutyl methyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose (e.g. available as Natrosol), sodium carboxymethyl cellulose, and colloidal silica such as finely ground Syloid (e.g. 244).
Solubilizing agents may also be included such as humectant polyols such propylene glycol, dipropylene glycol and hexylene glycol, cellosolves such as methyl cellosolve and ethyl cellosolve, vegetable oils and waxes containing at least about 12 carbons in a straight chain such as olive oil, castor oil and petrolatum and esters such as amyl acetate, ethyl acetate and benzyl benzoate.
It will be understood that, as is conventional, the oral preparations are to be sold or otherwise distributed in suitable labelled packages. Thus, a jar of mouthrinse will have a label describing it, in substance, as a mouthrinse or mouthwash and having directions for its use; and a toothpaste, cream or gel will usually be in a collapsible tube, typically aluminium, lined lead or plastic, or other squeeze, pump or pressurized dispenser for metering out the contents, having a label describing it, in substance, as a toothpaste, gel or dental cream.
Organic surface-active agents are used in the compositions of the present invention to achieve increased prophylactic action, assist in achieving thorough and complete dispersion of the active agent throughout the oral cavity, and render the instant compositions more cosmetically acceptable.
The organic surface-active material is preferably anionic, nonionic or ampholytic in nature which does not denature the antibody of the invention, and it is preferred to employ as the surface-active agent a detersive material which imparts to the composition detersive and foaming properties while not denaturing the antibody. Suitable examples of anionic surfactants are water-soluble salts of higher fatty acid monoglyceride monosulfates, such as the sodium salt of the monosulfated monoglyceride of hydrogenated coconut oil fatty acids, higher alkyl sulfates such as sodium lauryl sulfate, alkyl aryl sulfonates such as sodium dodecyl benzene sulfonate, higher alkylsulfo-acetates, higher fatty acid esters of 1,2-dihydroxy propane sulfonate, and the substantially saturated higher aliphatic acyl amides of lower aliphatic amino carboxylic acid compounds, such as those having 12 to 16 carbons in the fatty acid, alkyl or acyl radicals, and the like. Examples of the last mentioned amides are N-lauroyl sarcosine, and the sodium, potassium, and ethanolamine salts of N-lauroyl, N-myristoyl, or N-palmitoyl sarcosine which should be substantially free from soap or similar higher fatty acid material.
The use of these sarconite compounds in the oral compositions of the present invention is particularly advantageous since these materials exhibit a prolonged marked effect in the inhibition of acid formation in the oral cavity due to carbohydrates breakdown in addition to exerting some reduction in the solubility of tooth enamel in acid solutions. Examples of water-soluble nonionic surfactants suitable for use with antibodies are condensation products of ethylene oxide with various reactive hydrogen-containing compounds reactive therewith having long hydrophobic chains (e.g. aliphatic chains of about 12 to 20 carbon atoms), which condensation products (xe2x80x9cethoxamersxe2x80x9d) contain hydrophilic polyoxyethylene moieties, such as condensation products of poly(ethylene oxide) with fatty acids, fatty alcohols, fatty amides, polyhydric alcohols (e.g. sorbitan monostearate) and polypropyleneoxide (e.g. Pluronic materials).
Surface active agent is typically present in amount of about 0.1-5% by weight. It is noteworthy, that the surface active agent may assist in the dissolving of the antibody of the invention and thereby diminish the amount of solubilizing humectant needed.
Various other materials may be incorporated in the oral preparations of this invention such as whitening agents, preservatives, silicones, chlorophyll compounds and/or ammoniated material such as urea, diammonium phosphate, and mixtures thereof. These adjuvants, where present, are incorporated in the preparations in amounts which do not substantially adversely affect the properties and characteristics desired.
Any suitable flavoring or sweetening material may also be employed. Examples of suitable flavoring constituents are flavoring oils, e.g. oil of spearmint, peppermint, wintergreen, sassafras, clove, sage, eucalyptus, marjoram, cinnamon, lemon, and orange, and methyl salicylate. Suitable sweetening agents include sucrose, lactose, maltose, sorbitol, xylitol, sodium cyclamate, perillartine, AMP (aspartyl phenyl alanine, methyl ester), saccharine, and the like. Suitably, flavor and sweetening agents may each or together comprise from about 0.1% to 5% more of the preparation.
In the preferred practice of this invention an oral composition according to this invention such as mouthwash or dentifrice containing the composition of the present invention is preferably applied regularly to the gums and teeth, such as every day or every second or third day or preferably from 1 to 3 times daily, at a pH of about 4.5 to about 9, generally about 5.5 to about 8, preferably about 6 to 8, for at least 2 weeks up to 8 weeks or more up to a lifetime.
The compositions of this invention can be incorporated in lozenges, or in chewing gum or other products, e.g. by stirring into a warm gum base or coating the outer surface of a gum base, illustrative of which may be mentioned jelutong, rubber latex, vinylite resins, etc., desirably with conventional plasticizers or softeners, sugar or other sweeteners or such as glucose, sorbitol and the like.
Another important form of the invention is a composition for use in raising an immune response directed against P. gingivalis based on the four antigens and suitable adjuvant and/or carrier. This may be delivered via a number of routes, for example by nasal spray, orally or by injection to produce a specific immune response against the antigen thereby reducing colonisation of P. gingivalis and reducing virulence thereby preventing disease. As will be readily understood the composition may be based upon a recombinant antigen incorporated into an appropriate vector and expressed in a suitable transformed host (e.g. E. coli, Bacillus subtilis, Saccharomyces cerevisiae, COS cells, CHO cells and HeLa cells) containing the vector. Unlike whole P. gingivalis cells or other previously prepared antigens, the antigens described herein or peptides, oligopeptides or chimeric peptides are safe and effective antigens for the preparation of a vaccine for the prevention of P. gingivalis-associated periodontal disease. The antigenic protein, peptides, oligopeptides and chimeric peptides of the present invention, can be produced using recombinant DNA methods as illustrated herein, or can be synthesized chemically from the amino acid sequence disclosed in the present invention. Additionally, peptides can be produced from enzymatic or chemical cleavage of the purified antigens. Antigenic protein, peptides, and oligopeptides with immunogenic epitopes combined, can be used as immunogens in various vaccine formulations in the prevention of periodontal diseases. Additionally, according to the present invention, antigenic protein and related peptides or chimeras produced may be used to generate P. gingivalis antisera useful for passive immunization against periodontal disease and infections caused by P. gingivalis.
As opposed to use of the antigens themselves in eliciting an immune response this may be achieved by administration of a DNA molecule including a sequence encoding at one of the antigens or epitope containing fragment(s).
The present invention further provides the nucleotide sequence of the genes encoding the antigens, as well as the amino acid sequence deduced from the isolated genes. According to one particularly preferred embodiment of the present invention, using recombinant DNA techniques the genes encoding the antigens or gene fragments encoding one or more peptides or chimeras having immunogenic epitopes, is incorporated into an expression vector, and the recombinant vector is introduced into an appropriate host cell thereby directing the expression of these sequences in that particular host cell. The expression system, comprising the recombinant vector introduced into the host cell, can be used (a) to produce antigenic protein, related peptides, oligopeptides or chimeras which can be purified for use as an immunogen in vaccine formulations; (b) to produce antigenic protein, related peptides, oligopeptides and chimeras to be used as an antigen for diagnostic immunoassays or for generating P. gingivalis-specific antisera of therapeutic and/or diagnostic value; (c) or if the recombinant expression vector is a live virus such as vaccinia virus, the vector itself may be used as a live or inactivated vaccine preparation to be introduced into the host""s cells for expression of antigen or immunogenic peptides or oligopeptides or chimeric peptides; (d) for introduction into live attenuated bacterial cells or genetically engineered commensal intra-oral bacteria which are used to express antigenic protein, related peptides or oligopeptides or chimeras to vaccinate individuals; (e) or for introduction directly into an individual to immunize against the encoded and expressed antigenic protein, related peptides, or oligopeptides or chimeras. In particular the recombinant bacterial vaccine can be based on a commensal inhabitant of the human oral cavity or animal if the vaccine is to prevent periodontal disease in animals. The recombinant bacterial vaccine expressing antigen can be used to colonise the oral cavity, supragingival or subgingival plaque. The intra-oral bacterium can be isolated from the patient with periodontitis and genetically engineered to express the antigen, peptides or chimeras. The production of the P. gingivalis antigen within the oral cavity will not be toxic to the oral mucosal tissues. However, the expressed antigen will stimulate the mucosal-associated lymphoid tissues (MALT) to produce specific antibody to neuralise and reduce the virulence of P. gingivalis.